This invention relates to compounds which inhibit xcex2-amnyloid peptide release and/or its synthesis, and, accordingly, have utility in treating Alzheimer""s disease.
The following publications, patents and patent applications are cited in this application as superscript numbers:
1 Glenner, et al., xe2x80x9cAlzheimier""s Disease: Initial Report of the Purification and Characterization of a Novel Cerebrovascular Amyloid Proteinxe2x80x9d, Biochem. Biophys. Res. Commun., 120:885-890 (1984).
2 Glenner, et al., xe2x80x9cPolypeptide Marker for Alzheimner""s Disease and its Use for Diagnosisxe2x80x9d, U.S. Pat. No. 4,666,829 issued May 19, 1987.
3 Selkoe, xe2x80x9cThe Molecular Pathology of Alzheimer""s Diseasexe2x80x9d, Neuron, 6:487-498 (1991).
4 Goate, et al., xe2x80x9cSegregation of a Missense Mutation in the Amyloid Precursor Protein Gene with Familial Alzheimer""s Diseasexe2x80x9d, Nature, 349:704-706 (1990).
5 Chartier-Harlan, et al., xe2x80x9cEarly-Onset Alzheimer""s Disease Caused by Mutations at Codon 717 of the xcex2-Amyloid Precursor Proteing Genexe2x80x9d, Nature, 353:844-846 (1989).
6 Murrell, et al., xe2x80x9cA Mutation in the Amyloid Precursor Protein Associated with Hereditary Alzheimer""s Diseasexe2x80x9d, Science, 254:97-99 (1991).
7 Mullan, et al., xe2x80x9cA Pathogenic Mutation for Probable Alzheimer""s Disease in the APP Gene at the N-Terminus of xcex2-Amyloid, Nature Genet., 1:345-347 (1992).
8 Schenk, et al., xe2x80x9cMethods and Compositions for the Detection of Soluble xcex2-Amyloid Peptidexe2x80x9d, International Patent Application Publication No. WO 94/10569, published May 11, 1994.
9 Selkoe, xe2x80x9cAmyloid Protein and Alzheimer""s Diseasexe2x80x9d, Scientific American, pp. 2-8, November, 1991.
10 Yates, et al., xe2x80x9cN,N-Disubstituted Amino Acid Herbicidesxe2x80x9d, U.S. Pat. No. 3,598,859, issued Aug. 10, 1971.
11 Losse, et al., Tetrahedron, 27:1423-1434 (1971).
12 Citron, et al., xe2x80x9cMutation of the xcex2-Amyloid Precursor Protein in Familial Alzheimer""s Disease Increases xcex2-Protein Production, Nature, 360:672-674 (1992).
13 Hansen, et al., xe2x80x9cReexamination and Further Development of a Precise and Rapid Dye Method for Measuring Cell Growth/Cell Killxe2x80x9d, J. Immun. Meth., 119:203-210 (1989).
All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Alzheimer""s Disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms and course is currently known.
The brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Smaller numbers of these lesions in a more restrictive anatomical distribution are also found in the brains of most aged humans who do not have clinical AD. Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down""s Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCHWA-D). At present, a definitive diagnosis of AD usually requires observing the aforementioned lesions in the brain tissue of patients who have died with the disease or, rarely, in small biopsied samples of brain tissue taken during an invasive neurosurgical procedure.
The principal chemical constituent of the amyloid plaques and vascular amyloid deposits (amyloid angiopathy) characteristic of AD and the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 39-43 amino acids designated the xcex2-amyloid peptide (xcex2AP) or sometimes Axcex2, Axcex2P or xcex2/A4. xcex2-Amyloid peptide was first purified and a partial amino acid sequence was provided by Glenner, et al.1 The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,8292.
Molecular biological and protein chemical analyses have shown that the xcex2-amyloid peptide is a small fragment of a much larger precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans. Knowledge of the structure of the gene encoding the APP has demonstrated that xcex2-amyloid peptide arises as a peptide fragment that is cleaved from APP by protease enzyme(s). The precise biochemical mechanism by which the xcex2-amyloid peptide fragment is cleaved from APP and subsequently deposited as amyloid plaques in the cerebral tissue and in the walls of the cerebral and meningeal blood vessels is currently unknown.
Several lines of evidence indicate that progressive cerebral deposition of xcex2-amyloid peptide plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades. See, for example, Selkoe3. The most important line of evidence is the discovery that missense DNA mutations at amino acid 717 of the 770-amino acid isoform of APP can be found in affected members but not unaffected members of several families with a genetically determined (familial) form of AD (Goate, et al.4; Chartier Harlan, et al.5; and Murrell, et al.6) and is referred to as the Swedish variant. A double mutation changing lysine595-methionine596 to asparagine595-leucine596 (with reference to the 695 isoform) found in a Swedish family was reported in 1992 (Mullan, et al.7). Genetic linkage analyses have demonstrated that these mutations, as well as certain other mutations in the APP gene, are the specific molecular cause of AD in the affected members of such families. In addition, a mutation at amino acid 693 of the 770-amino acid isoform of APP has been identified as the cause of the xcex2-amyloid peptide deposition disease, HCHWA-D, and a change from alanine to glycine at amino acid 692 appears to cause a phenotype that resembles AD is some patients but HCHWA-D in others. The discovery of these and other mutations in APP in genetically based cases of AD prove that alteration of APP and subsequent deposition of its xcex2-amyloid peptide fragment can cause AD.
Despite the progress which has been made in understanding the underlying mechanisms of AD and other xcex2-amyloid peptide related diseases, there remains a need to develop methods and compositions for treatment of the disease(s). Ideally, the treatment methods would advantageously be based on drugs which are capable of inhibiting xcex2-amyloid peptide release and/or its synthesis in vivo.
This invention is directed to the discovery of a class of compounds which inhibit xcex2-amyloid peptide release and/or its synthesis and, therefore, are useful in the prevention of AD in patients susceptible to AD and/or in the treatment of patients with AD in order to inhibit further deterioration in their condition. The class of compounds having the described properties are defined by formula I below: 
wherein
R1 is selected from the group consisting of:
(a) a substituted phenyl group of formula II: 
xe2x80x83wherein
Rc is selected from the group consisting of acyl, alky, alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and wherein Rb and Rc are fused to form a heteroaryl or heterocyclic ring with the phenyl ring wherein the heteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from 1 to 3 are heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur;
Rb and Rbxe2x80x2 are independently selected from the group consisting of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy with the proviso that Rb, Rbxe2x80x2 and Rc are not all hydrogen and with the further proviso that when Rc is hydrogen, then neither Rb nor Rbxe2x80x2 are hydrogen;
(b) 2-naphthyl; and
(c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1 to 5 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy;
R2 is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms; and
R3 is selected from the group consisting of:
(a) xe2x80x94Y(CH2)nCHR4R5 wherein n is an integer of from 0 to 2, Y is selected from the group consisting of oxygen and sulfur, R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl optionally substituted with from 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy, heteroaryl optionally substituted with from 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy, and where R4 and R5 are joined to form a cycloalkyl group, a cycloalkenyl group, or a heterocyclic group;
(b) xe2x80x94ONxe2x95x90C(NH2)R6 where R6 is selected from the group consisting of alkyl, aryl, cycloalkyl, and heteroaryl;
(c) xe2x80x94O(CH2)pC(O)OR7 wherein p is an integer of from 1 to 2 and R7 is alkyl;
(d) xe2x80x94NR8R9 wherein R8 and R9 are joined to form a pyrrolyl group; and
pharmaceutically acceptable salts thereof
with the provisos that
1. when R1 is the substituted phenyl group of formula II above, Rbxe2x80x2 is hydrogen, Rb and Rc are chloro, and R2 is methyl, then R3 is not xe2x80x94OCH(CH3)-xcfx86;
2. when R1 is the substituted phenyl group of formula II above, when Rbxe2x80x2 is hydrogen, Rb and Rc are chloro, and R3 is xe2x80x94OCH2CH3 then R2 is not hydrogen;
3. when R1 is the substituted phenyl group of formula II above, Rbxe2x80x2 is hydrogen, Rb and Rc are chloro, and R3 is xe2x80x94OCH2CH(CH3)2 then R2 is not xe2x80x94CH(CH3)CH2CH3; and
4. when R1 is N-methylindol-5-yl and R2 is methyl, then R3 is not xe2x80x94OCH2CH3.
Surprisingly, any substituents at the 2 and/or 6 positions or substituents at the 3, 4 and/or 5 positions, other than those specifically specified above, eliminate the ability of the resulting compounds to inhibit xcex2-amyloid peptide release and/or its synthesis.
Accordingly, in one of its method aspects, this invention is directed to a method for inhibiting xcex2-amyloid peptide release and/or its synthesis in a cell which method comprises administering to such a cell an amount of a compound or a mixture of compounds of formula I above effective in inhibiting the cellular release and/or synthesis of xcex2-amyloid peptide.
Because the in vivo generation of xcex2-amyloid peptide is associated with the pathogenesis of AD8,9, the compounds of formula I can also be employed in conjunction with a pharmaceutical composition to prophylactically and/or therapeutically prevent and/or treat AD. Accordingly, in another of its method aspects, this invention is directed to a prophylactic method for preventing the onset of AD in a patient at risk for developing AD which method comprises administering to said patient a pharmaceutical composition comprising a pharmaceutically inert carrier and an effective amount of a compound or a mixture of compounds of formula I above.
In yet another of its method aspects, this invention is directed to a therapeutic method for treating a patient with AD in order to inhibit further deterioration in the condition of that patient which method comprises administering to said patient a pharmaceutical composition comprising a pharmaceutically inert carrier and an effective amount of a compound or a mixture of compounds of formula I above.
In formula I above, R1 substituted phenyls are preferably 4-substituted, 3,5-disubstituted or 3,4-disubstituted phenyl substituents wherein the substituents at the 3 and/or 5 positions are defined by Rb, Rbxe2x80x2 as above and the substituent at the 4 position is defined by Rc as above. Particularly preferred 3,5-disubstituted phenyls include, by way of example, 3,5-dichlorophenyl, 3,5-difluorophenyl, 3,5-di(trifluoromethyl)phenyl, 3,5-dimethoxyphenyl, and the like. Particularly, preferred 3,4-disubstituted phenyls include, by way of example, 3,4-dichlorophenyl, 3,4-difluorophenyl, 3-(trifluoromethyl)-4-chlorophenyl, 3-chloro-4-cyanophenyl, 3-chloro-4-iodophenyl, 3,4-methylenedioxyphenyl, 3,4-ethylenedioxyphenyl, and the like. Particularly preferred 4-substituted phenyls include, by way of example, 4-azidophenyl, 4-bromophenyl, 4-chlorophenyl, 4-cyanophenyl, 4-ethylphenyl, 4-fluorophenyl, 4-iodophenyl, 4-(phenylcarbonyl)phenyl, 4-(1-ethoxy)ethylphenyl, 4-(ethoxycarbonyl)phenyl, and the like.
Other preferred R1 substituents include, by way of example, 2-naphthyl, 2-methylquinolin-6-yl, benzothiazol-6-yl, 5-indolyl, and the like.
Preferably R2 is selected from the group consisting of alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms. Particularly preferred R2 substituents include, by way of example, methyl, ethyl, n-propyl, iso-butyl, and the like.
Preferred R3 substituents include methoxy, ethoxy, iso-propoxy, n-propoxy, n-butoxy, iso-butoxy, cyclopentoxy, allyloxy, 4-methylpentoxy, xe2x80x94Oxe2x80x94CH2-(2,2-dimethyl-1,3-dioxolan-4-yl), xe2x80x94Oxe2x80x94CH2-cyclohexyl, xe2x80x94Oxe2x80x94CH2-(3-tetrahydrofuranyl), xe2x80x94Oxe2x80x94CH2xe2x80x94C(O)O-tert-butyl, xe2x80x94Oxe2x80x94CH2xe2x80x94C(CH3)3, xe2x80x94Oxe2x80x94CH2-xcfx86, xe2x80x94OCH2CH(CH2CH3)2, xe2x80x94O(CH2)3CH(CH3)2, xe2x80x94ONxe2x95x90C(NH2)xcfx86, xe2x80x94ONxe2x95x90C(NH2)CH3, xe2x80x94ONxe2x95x90C(NH2)CH2CH3, xe2x80x94ONxe2x95x90C(NH2)CH2CH2CH3, xe2x80x94ONxe2x95x90C(NH2)-cyclopropyl, xe2x80x94ONxe2x95x90C(NH2)xe2x80x94CH2-cyclopropyl, xe2x80x94ONxe2x95x90C(NH2)-cyclopentyl, xe2x80x94ONxe2x95x90C(NH2)CH2CH(CH3)2, and the like.
This invention also provides for novel pharmaceutical compositions comprising a pharmaceutically inert carrier and a compound of the formula I above.
Particularly preferred compounds for use in the methods and compositions of this invention include, by way of example, the following wherein the stereochemistry of the R2 group (where appropriate) is derived from the L-amino acid:
N-(3,4-dichlorophenyl)alanine ethyl ester;
N-(3-trifluoromethyl-4-chlorophenyl)alanine ethyl ester;
N-(3,5-dichlorophenyl)alanine ethyl ester;
N-(3,4-difluorophenyl)alanine ethyl ester;
N-(3,4-dichlorophenyl)alanine benzyl ester;
N-(3,4-dichlorophenyl)alanine iso-butyl ester;
N-(3,4-dichlorophenyl)alanine iso-propyl ester;
N-(3,4-dichlorophenyl)alanine n-butyl ester;
N-(3,4-dichlorophenyl)alanine methyl ester;
N-(3,4-dichlorophenyl)alanine cyclopentyl ester;
N-(3,4-dichlorophenyl)alanine n-propyl ester;
N-(3,4-dichlorophenyl)alanine allyl ester;
N-(3,4-dichlorophenyl)alanine 4-methylpentyl ester;
N-(3,4-dichlorophenyl)alanine 2,2-dimethyl-1,3-dioxolane-4-methyl ester;
N-(3,4-dichlorophenyl)alanine cyclohexylmethyl ester;
N-(3,4-dichlorophenyl)alanine tert-butoxycarbonylmethyl ester;
N-(3,4-dichlorophenyl)leucine iso-butyl ester;
2-[N-(3,4-dichlorophenyl)amino]pentanoic acid iso-butyl ester;
N-(4-cyanophenyl)alanine iso-butyl ester;
N-(3-chloro-4-cyanophenyl)alanine iso-butyl ester;
N-(3,4-dichlorophenyl)alanine tetrahydrofuran-3-yl-methyl ester;
N-(3-chloro-4-iodophenyl)alanine iso-butyl ester;
2-[N-(3,4-dichlorophenyl)amino]butanoic acid isobutyl ester;
N-(4-chlorophenyl)alanine iso-butyl ester;
N-(3,5-dichlorophenyl)alanine iso-butyl ester;
N-(4-ethylphenyl)alanine methyl ester;
N-[4-(1-ethoxy)ethylphenyl]alanine methyl ester;
N-(3,4-dichlorophenyl)alanine 2,2-dimethylpropyl ester;
N-(3,4-dichlorophenyl)glycine iso-butyl ester;
N-(3,4-dichlorophenyl)alanine 2-ethylbutyl ester;
N-(3-chloro-4-iodophenyl)alanine iso-butyl ester;
N-(4-azidophenyl)alanine iso-butyl ester;
N-[(4-phenylcarbonyl)phenyl]alanine iso-butyl ester;
N-(3,5-difluorophenyl)alanine iso-butyl ester;
N-(3,4-dichlorophenyl)alanine O-acylacetamidoxime ester,
N-(3,4-dichlorophenyl)alanine pyrrolyl amide;
N-(3,4-dichlorophenyl)alanine O-acylpropionamideoxime ester;
N-(3,4-dichlorophenyl)alanine O-acylbutyramideoxime ester;
2-[N-(naphth-2-yl)amino]butanoic acid ethyl ester;
N-(naphth-2-yl)alanine iso-butyl ester;
N-(2-methylquinolin-6-yl)alanine iso-butyl ester;
N-(3,4-ethylenedioxyphenyl)alanine iso-butyl ester;
N-(3,4-methylenedioxyphenyl)alanine iso-butyl ester;
N-(naphth-2-yl)alanine methyl ester;
N-(benzothiazol-6-yl)alanine ethyl ester;
N-(indol-5-yl)alanine iso-butyl ester;
N-(naphth-2-yl)alanine O-acylacetamidoxime ester;
N-(2-naphthyl)alanine ethyl ester;
N-(4-ethoxycarbonylphenyl)alanine iso-butyl ester;
N-(3,5-di(trifluoromethyl)phenyl)alanine iso-butyl ester;
N-(3,5-dimethoxyphenyl)alanine iso-butyl ester;
N-(2-napthyl)alanine O-acylpropionamidoxime ester;
N-(2-napthyl)alanine O-acylbutyramidoxime ester;
N-(2-napthyl)alanine O-acylisovaleramidoxie ester;
N-(2-napthyl)alanine O-acylbenzamidoxime ester;
N-(2-napthyl)alanine O-acylcyclopropanecarboxamidoxime ester;
N-(2-napthyl)alanine O-acylcyclopropylacetamidoxime ester; and
N-(2-napthyl)alanine O-acylcyclopentanecarboxamidoxime ester.
Still further, this invention provides for novel compounds of the formula III: 
wherein
R1 is selected from the group consisting of:
(a) a substituted phenyl group of formula II: 
xe2x80x83wherein
Rc is selected from the group consisting of acyl, alkyl, alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and where Rb and Rc are fused to form a heteroaryl or heterocyclic ring with the phenyl ring wherein the heteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from 1 to 3 are heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur;
Rb and Rbxe2x80x2 are independently selected from the group consisting of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy with the proviso that Rb, Rbxe2x80x2 and Rc are not all hydrogen and with the further proviso that when Rc is hydrogen, then neither Rb nor Rbxe2x80x2 are hydrogen;
(b) 2-naphthyl; and
(c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1 to 5 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy;
R2 is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms; and
R3 is selected from the group consisting of:
(a) xe2x80x94Y(CH2)nCHR4R5 wherein n is an integer of from 0 to 2, Y is selected from the group consisting of oxygen and sulfur, R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl optionally substituted with from 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy, heteroaryl optionally substituted with from 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy, and where R4 and R5 are joined to form a cycloalkyl group, a cycloalkenyl group or a heterocyclic group;
(b) xe2x80x94ONxe2x95x90C(NH2)R6 where R6 is selected from the group consisting of alkyl, aryl, cycloalkyl, and heteroaryl;
(c) xe2x80x94O(CH2)pC(O)OR7 wherein p is an integer of from 1 to 2 and R8 is alkyl; and
(d) xe2x80x94NR8R9 wherein R8 and R9 are joined to form a pyrrolyl group;
and pharmaceutically acceptable salts thereof
with the proviso excluding the following compounds:
1. when R1 is the substituted phenyl group of formula II above, Rbxe2x80x2 is hydrogen, Rb and Rc are chloro, and R2 is methyl, then R3 is not xe2x80x94OCH(CH3)-xcfx86;
2. when R1 is the substituted phenyl group of formula II above, when Rbxe2x80x2 is hydrogen, Rb and Rc are chloro, and R3 is xe2x80x94OCH2CH3 then R2 is not hydrogen;
3. when R1 is the substituted phenyl group of formula II above, Rbxe2x80x2 is hydrogen, Rb and Rc are chloro, and R3 is xe2x80x94OCH2CH(CH3)2 then R2 is not xe2x80x94CH(CH3)CH2CH3; and
4. when R1 is N-methylindol-5-yl and R2 is methyl, then R3 is not xe2x80x94OCH2CH3;
and still with further proviso excluding the following known compounds: N-(4-chlorophenyl)alanine ethyl ester; N-(3,4-dichlorophenyl)alanine ethyl ester; N-(3,5-dichlorophenyl)alanine ethyl ester; N-(4-n-butylphenyl)alanine ethyl ester; N-(3,4-dinitrophenyl)alanine ethyl ester; N-(4-chlorophenyl)glycine heptenyl ester; N-(4-methylphenyl)glycine butyl ester; N-(3-nitrophenyl)glycine decyl ester; N-(3,4-difluorophenyl)alanine methyl ester; N-(3,4difluorophenyl)alanine ethyl ester; N-(3,4-difluorophenyl)alanine iso-propyl ester; N-(4-fluorophenyl)alanine ethyl ester; N-(3-chloro-4-fluorophenyl)alanine methyl ester; N-(3-chloro-4-fluorophenyl)alanine ethyl ester; and N-(3-chloro-4-fluorophenyl)alanine iso-propyl ester.
Preferred compounds of formula I above include those set forth in Formula IV below:
Other preferred compounds of formula I include those set forth in the following formula V:
As above, this invention relates to compounds which inhibit xcex2-amyloid peptide release and/or its synthesis, and, accordingly, have utility in treating Alzheimer""s disease. However, prior to describing this invention in further detail, the following terms will first be defined.
Definitions
The term xe2x80x9cxcex2-amyloid peptidexe2x80x9d refers to a 39-43 amino acid peptide having a molecular weight of about 4.2 kD, which peptide is substantially homologous to the form of the protein described by Glenner, et al.1 including mutations and post-translational modifications of the normal xcex2-amyloid peptide. In whatever form, the xcex2-amyloid peptide is approximately a 39-43 amino acid fragment of a large membrane-spanning glycoprotein, referred to as the xcex2-amyloid precursor protein (APP). Its 43-amino acid sequence is:
1
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
11
Glu Val His His Gln Lys Leu Val Phe Phe
21
Ala Glu Asp Val Gly Ser Asn Lys Gly Ala
31
Ile Ile Gly Leu Met Val Gly Gly Val Val
41
Ile Ala Thr (SEQ ID NO: 1)
or a sequence which is substantially homologous thereto.
xe2x80x9cAlkylxe2x80x9d refers to monovalent alkyl groups preferably having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, and the like.
xe2x80x9cAlkylenexe2x80x9d refers to divalent alkylene groups preferably having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methylene (xe2x80x94CH2xe2x80x94), ethylene (xe2x80x94CH2CH2xe2x80x94), the propylene isomers (e.g., xe2x80x94CH2CH2CH2xe2x80x94 and xe2x80x94CH(CH3)CH2xe2x80x94) and the like.
xe2x80x9cAlkarylxe2x80x9d refers to -alkylene-aryl groups preferably having from 1 to 10 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.
xe2x80x9cAlkoxyxe2x80x9d refers to the group xe2x80x9calkyl-Oxe2x80x94xe2x80x9d where alkyl is as defined herein. Preferred alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
xe2x80x9cAlkoxycarbonylxe2x80x9d refers to the group xe2x80x9calkyl-Oxe2x80x94C(O)xe2x80x94xe2x80x9d wherein alkyl is as defined herein. Such groups include, by way of example, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, iso-propoxycarbonyl, n-butoxycarbonyl, tert-butoxyfarbonyl, sec-butoxycarbonyl, n-pentoxycarbonyl, n-hexoxycarbonyl, and the like.
xe2x80x9cAlkylalkoxyxe2x80x9d refers to the group xe2x80x9c-alklene-O-alkylxe2x80x9d wherein alkylene and alkoxy are as defined herein. Such groups include, by way of example, methyl methoxy (xe2x80x94CH2OCH3), ethyl methoxy (xe2x80x94CH2CH2OCH3), n-propyl-iso-propoxy (xe2x80x94CH2CH2CH2OCH(CH3)2), methyl-tert-butoxy (xe2x80x94CH2xe2x80x94Oxe2x80x94C(CH3)3) and the like.
xe2x80x9cAlkylthioalkoxyxe2x80x9d refers to the group xe2x80x9c-alkylene-S-alkylxe2x80x9d wherein alkylene and alkoxy are as defined herein. Such groups include, by way of example, methylthiomethoxy (xe2x80x94CH2SCH3), ethylthiomethoxy (xe2x80x94CH2CH2SCH3), n-propyl-iso-thiopropoxy (xe2x80x94CH2CH2CH2SCH(CH3)2), methyl-tert-thiobutoxy (xe2x80x94CH2SC(CH3)3) and the like.
xe2x80x9cAlkenylxe2x80x9d refers to alkenyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkenyl unsaturation. Preferred alkenyl groups include ethenyl (xe2x80x94CHxe2x95x90CH2), n-propenyl (xe2x80x94CH2CHxe2x95x90CH2), iso-propenyl (xe2x80x94C(CH3)xe2x95x90CH2), but-2-enyl (xe2x80x94CH2CHxe2x95x90CHCH3) and the like.
xe2x80x9cAlkynylxe2x80x9d refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkynyl unsaturation. Preferred alkynyl groups include ethynyl (xe2x80x94Cxe2x89xa1CH), propargyl (xe2x80x94CH2Cxe2x89xa1CH) and the like.
xe2x80x9cAcylxe2x80x9d refers to the groups alkyl-C(O)xe2x80x94, aryl-C(O)xe2x80x94, and heteroaryl-C(O)xe2x80x94 where alkyl, aryl and heteroaryl are as defined herein.
xe2x80x9cAcylaminoxe2x80x9d refers to the group xe2x80x94C(O)NRR where each R is independently hydrogen or alkyl where alkyl is as defined herein.
xe2x80x9cAminoacylxe2x80x9d refers to the group xe2x80x94NRC(O)R where each R is independently hydrogen or alkyl where alkyl is as defined herein.
xe2x80x9cAcyloxyxe2x80x9d refers to the groups alkyl-C(O)Oxe2x80x94, aryl-C(O)Oxe2x80x94, heteroaryl-C(O)Oxe2x80x94, and heterocyclic-C(O)Oxe2x80x94 where alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
xe2x80x9cArylxe2x80x9d refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like.
Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 3 substituents selected from the group consisting of hydroxy, acyl, acyloxy, alkyl, alkoxy, alkenyl, alkynyl, amino, aminoacyl, aryl, aryloxy, carboxyl, alkoxycarbonyl, acylamino, cyano, halo, nitro, heteroaryl, trihalomethyl and the like. Preferred substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.
xe2x80x9cAryloxyxe2x80x9d refers to the group aryl-Oxe2x80x94 wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
xe2x80x9cCycloalkylxe2x80x9d refers to cyclic alkyl groups of from 3 to 10 carbon atoms having a single cyclic ring or multiple condensed rings which can be optionally substituted with from 1 to 3 alkyl groups. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
xe2x80x9cCycloalkenylxe2x80x9d refers to cyclic alkenyl groups of from 4 to 8 carbon atoms having a single cyclic ring and at least one point of internal unsaturation which can be optionally substituted with from 1 to 3 alkyl groups. Examples of suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.
xe2x80x9cHaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d refers to fluoro, chloro, bromo and iodo and preferably is either chloro or fluoro.
xe2x80x9cHeteroarylxe2x80x9d refers to a monovalent aromatic group of from 2 to 8 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within the ring.
Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thioalkoxy, thioaryloxy and the like. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl and furyl.
xe2x80x9cHeterocyclexe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur or oxygen within the ring.
Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro, heteroaryl, thioalkoxy, thioaryloxy and the like. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzoftu or quinuclidinyl). Preferred heterocycles include piperidinyl, pyrrolidinyl and tetrahydrofiiryl.
Examples of heterocycles and heteroaryls include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline and the like.
In the compounds of formula I, Rb and Rc can be fused to form a heteroaryl or heterocyclic ring with the phenyl ring. Fusion in this manner results in a fused bicyclic ring structure of the formula: 
where Rbxe2x80x2 is as defined above and A is the fused heteroaryl or heterocyclic group containing from 3 to 8 atoms of which from 1 to 3 are heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur wherein the two atoms of the phenyl ring are included in the total atoms present in the heteroaryl or heterocyclic group. Examples of such fused ring systems include, for instance, indol-5-yl, indol-6-yl, thionaphthen-5-yl, thionaphthen-6-yl, isothionaphthen-5-yl, isothionaphthen-6-yl, indoxazin-5-yl, indoxazin-6-yl, benzoxazol-5-yl, benzoxazol-6-yl, anthranil-5-yl, anthranil-6-yl, quinolin-6-yl, quinolin-7-yl, isoquinolin-6-yl, isoquinolin-7-yl, cinnolin-6-yl, cinnolin-7-yl, quinazolin-6-yl, quinazolin-7-yl, benzofuran-5-yl, benzofuran-6-yl, isobenzofuran-5-yl, isobenzofuran-6-yl, and the like.
xe2x80x9cThiolxe2x80x9d refers to the group xe2x80x94SH.
xe2x80x9cThioalkoxyxe2x80x9d refers to the group xe2x80x94S-alkyl.
xe2x80x9cThioaryloxyxe2x80x9d refers to the group aryl-Sxe2x80x94 wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
xe2x80x9cThioheteroaryloxyxe2x80x9d refers to the group heteroaryl-Sxe2x80x94 wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.
xe2x80x9cPharmaceutically acceptable saltxe2x80x9d refers to pharmaceutically acceptable salts of a compound of Formula I which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
Compound Preparation
The compounds of formula I above are readily prepared via several divergent synthetic routes with the particular route selected relative to the ease of compound preparation, the commercial availability of starting materials, and the like.
A first synthetic method involves conventional coupling of a halo acetic acid with a primary amine to form the amino acid followed by conventional esterification as shown in reaction (1) below: 
wherein R1, R2, R3 are as defined above and X is a halo group such as chloro or bromo. Alternatively, leaving groups other than halo may be employed such as triflate, tosylate, mesylate and the like. Additionally, a suitable ester of 1 may be employed in this reaction.
The first step of reaction (1) involves coupling of a suitable haloacetic acid derivative 1 with a primary aryl/heteroarylamine 2 under conditions which provide for amino acid 3. This reaction is described by, for example, Yates, et al.10 and proceeds by combining approximately stoichiometric equivalents of haloacetic acid 1 with primary aryl/heteroarylamine 2 in a suitable inert diluent such as water, dimethylsulfoxide (DMSO) and the like. The reaction employs an excess of a suitable base such as sodium bicarbonate, sodium hydroxide, etc. to scavenge the acid generated by the reaction. The reaction is preferably conducted at from about 25xc2x0 C. to about 100xc2x0 C. until reaction completion which typically occurs within 1 to about 24 hours. This reaction is further described in U.S. Pat. No. 3,598,859, which is incorporated herein by reference in its entirety. Upon reaction completion, N-aryl/N-heteroaryl amino acid 3 is recovered by conventional methods including precipitation, chromatography, filtration and the like.
N-aryl/N-heteroaryl amino acid 3 is next esterified with alcohol 4 by conventional esterificafion conditions to provide for the esterified N-aryl/N-heteroaryl amino acid 5 which is a compound of formula I. For example, esterification procedures for R3 groups containing an ester group can be achieved by using the methods of Losse, et al.11 If desired, the esterification reaction can optionally be conducted on haloacetic acid 1 prior to amination with aryl/heteroarylamine 2.
In reaction (1), each of the reagents (haloacetic acid 1, primary aryl/heteroarylamine 2 and alcohol 3) are well known in the art with a plurality of each being commercially available.
In an alternative embodiment, the R1 group can be coupled to an alanine ester (or other suitable amino acid ester) by conventional N-arylation. For example, a stoichiometric equivalent or slight excess of the amino acid ester can be dissolved in a suitable diluent such as DMSO and coupled with a haloaryl compound, Xxe2x80x94R1 where X is a halo group such as fluoro, chloro or bromo and R1 is as defined above. The reaction is conducted in the presence of an excess of base such as sodium hydroxide to scavenge the acid generated by the reaction. The reaction typically proceeds at from 15xc2x0 C. to about 250xc2x0 C. and is complete in about 1 to 24 hours. Upon reaction completion, N-aryl amino acid ester is recovered by conventional methods including chromatography, filtration and the like.
In still another alternative embodiment, the esterified amino acids of formula I above can be prepared by reductive amination of a 2-oxocarboxylic acid ester (such as a pyruvate ester) ester in the manner illustrated in Reaction (2) below: 
wherein R1, R2, R3 are as defined above.
In reaction (2), approximately stoichiometric equivalents of pyruvate ester 6 and arylamine 2 are combined in an inert diluent such as methanol, ethanol and the like and the reaction solution treated under conditions which provide for imine formation (not shown). The imine formed is then reduced under conventional conditions by a suitable reducing agent such as sodium cyanoborohydride, H2/palladium on carbon and the like to form the N-aryl amino acid ester 5. In a particularly preferred embodiment, the reducing agent is H2/palladium on carbon which is incorporated into the initial reaction medium which permits imine reduction in situ in a one pot procedure to provide for the N-aryl amino acid ester 5.
The reaction is preferably conducted at from about 20xc2x0 C. to about 80xc2x0 C. at a pressure of from 1 to 10 atmospheres until reaction completion which typically occurs within 1 to about 24 hours. Upon reaction completion, N-aryl amino acid ester 5 is recovered by conventional methods including chromatography, filtration and the like.
A further embodiment for preparing the compounds of formula I above includes aromatic nucleophilic substitution of fluorobenzenes by the amine group of an amino acid as set forth in the Examples below.
In still a further embodiment, conventional transesterification techniques can be used to prepare a variety of different ester groups on the N-aryl amino acid esters 5. Numerous techniques are known in the art to effect transesterification and each technique merely replaces the xe2x80x94OR3 group on the ester of the N-aryl amino acid ester 5 with a different xe2x80x94OR3 group derived from the corresponding alcohol (i.e., HOR3) and, in some cases, a catalyst such as titanium (IV) iso-propoxide is used to facilitate reaction completion. In one technique, the alcohol HOR3 is first treated with sodium hydride in a suitable diluent such as toluene to form the corresponding Na+ OR3 which is then employed to effect transesterification with the N-aryl amino acid ester 5. The efficiency of this technique makes it particularly useful with high boiling and/or expensive alcohols.
In another transesterification technique, the N-aryl amino acid ester 5 to be transesterified is placed in a large excess of the alcohol which effects transesterification. A catalytic amount of sodium hydride is then added and the reaction proceeds quickly under conventional conditions to provide the desired transesterified product. Because this protocol requires the use of a large excess of alcohol, this procedure is particularly useful when the alcohol is inexpensive.
Transesterification provides a facile means to provide for a multiplicity of R3 substituents on the compounds of formula I above. In all cases, the alcohols employed to effect transesterification are well known in the art with a significant number being commercially available.
Other methods for preparing the esters of this invention include, by way of example, first hydrolyzing the ester to the free acid followed by O-alkylation with a halo-R3 group in the presence of a base such as potassium carbonate.
Still other methods for the preparation of esters are provided in the examples below.
Methods for the preparation of O-acyloxime esters include transesterification of the trichlorophenyl ester of a carboxylic acid with an oxime, and coupling of a carboxylic acid and an oxime using a carbodiimide coupling reagent. Similarly, methods for the preparation of pyrrole amides include conventional amidation techniques of the corresponding acid and pyrrole.
In these synthetic methods, the starting materials can contain a chiral center (e.g., alanine) and, when a racemic starting material is employed, the resulting product is a mixture of R,S enantiomers. Alternatively, a chiral isomer of the starting material can be employed and, if the reaction protocol employed does not racemize this starting material, a chiral product is obtained. Such reaction protocols can involve inversion of the chiral center during synthesis.
Accordingly, unless otherwise indicated, the products of this invention are a mixture of R,S enantiomers. Preferably, however, when a chiral product is desired, the chiral product corresponds to the L-amino acid derivative. Alternatively, chiral products can be obtained via purification techniques which separates enantiomers from a R,S mixture to provide for one or the other stereoisomer. Such techniques are well known in the art.
Pharmaceutical Formulations
When employed as pharmaceuticals, the compounds of formula I are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds of formula I above associated with pharmaceutically acceptable carriers. In making the compositions of this invention, the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.
Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
The compositions are preferably formulated in a unit dosage form, each dosage containing from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient. The term xe2x80x9cunit dosage formsxe2x80x9d refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Preferably, the compound of formula I above is employed at no more than about 20 weight percent of the pharmaceutical composition, more preferably no more than about 15 weight percent, with the balance being pharmaceutically inert carrier(s).
The active compound is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It, will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient""s symptoms, and the like.
For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.